The Experts below are selected from a list of 7428 Experts worldwide ranked by ideXlab platform
Haobo Jiang - One of the best experts on this subject based on the ideXlab platform.
-
inhibition of immune pathway initiating hemolymph protease 14 by manduca sexta Serpin 12 a conserved mechanism for the regulation of melanization and toll activation in insects
Insect Biochemistry and Molecular Biology, 2020Co-Authors: Yang Wang, Michael R. Kanost, Xiaolong Cao, Fan Yang, Rudan Huang, Susan M Paskewitz, Steve Hartson, Haobo JiangAbstract:A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by Serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta Serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with Serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against Serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal β-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with Serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by Serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae Serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a Serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.
-
The Manduca sexta Serpinome: Analysis of Serpin genes and proteins in the tobacco hornworm.
Insect biochemistry and molecular biology, 2018Co-Authors: Jayne M. Christen, Neal T Dittmer, Xiaolong Cao, Xiufeng Zhang, Haobo JiangAbstract:Members of the Serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the Serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 Serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (Serpin-1, Serpin-15, and Serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl-terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twenty-eight of the 32 M. sexta Serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of Serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the Serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these Serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.
-
manduca sexta Serpin 12 controls the prophenoloxidase activation system in larval hemolymph
Insect Biochemistry and Molecular Biology, 2018Co-Authors: Fan Yang, Michael R. Kanost, Xiaolong Cao, Yang Wang, Niranji Sumathipala, Haobo JiangAbstract:Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the Serpin superfamily. The enzyme system also leads to proteolytic processing of a Spatzle precursor. Binding of Spatzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spatzle to induce immunity-related genes. Here we provide evidence that M. sexta Serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other Serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to Serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The Serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the Serpin-12 core domain (Serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the Serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both Serpin-12 and HP14, suggesting that a covalent Serpin-protease complex formed when Serpin-12 inhibited HP14. Together, these data suggest that M. sexta Serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.
-
identification of plasma proteases inhibited by manduca sexta Serpin 4 and Serpin 5 and their association with components of the prophenol oxidase activation pathway
Journal of Biological Chemistry, 2005Co-Authors: Youre Tong, Haobo Jiang, Michael R KanosAbstract:One innate immune response pathway of insects is a serine protease cascade that activates prophenol oxidase (pro-PO) in plasma. However, details of this pathway are not well understood, including the number and order of proteases involved. Protease inhibitors from the Serpin superfamily appear to regulate the proteases in the pathway. Manduca sexta Serpin-4 and Serpin-5 suppress pro-PO activation in plasma, apparently by inhibiting proteases upstream of the direct activator of pro-PO. To identify plasma proteases inhibited by these Serpins, we used immunoaffinity chromatography with Serpin antibodies to isolate Serpin-protease complexes that formed after activation of the cascade by exposure of plasma to bacteria or lipopolysaccharide. Covalent complexes of Serpin-4 with hemolymph proteases HP-1 and HP-6 appeared in plasma activated by Gram-positive or Gram-negative bacteria, whereas Serpin-4 complexes with HP-21 and two unidentified proteases were unique to plasma treated with Gram-positive bacteria. HP-1 and HP-6 were also identified as target proteases of Serpin-5, forming covalent complexes after bacterial activation of the cascade. These results suggest that HP-1 and HP-6 may be components of the pro-PO activation pathway, which are activated in response to infection and regulated by Serpin-4 and Serpin-5. HP-21 and two unidentified proteases may participate in a Gram-positive bacteria-specific branch of the pathway. Several plasma proteins that co-purified with Serpin-protease complexes, most notably immulectins and serine protease homologs, are known to be components of the pro-PO activation pathway. Our results suggest that after activation by exposure to bacteria, components of the pro-PO pathway associate to form a large noncovalent complex, which localizes the melanization reaction to the surface of invading microorganisms.
-
manduca sexta Serpin 6 regulates immune serine proteinases pap 3 and hp8 cdna cloning protein expression inhibition kinetics and function elucidation
Journal of Biological Chemistry, 2005Co-Authors: Zhen Zou, Haobo JiangAbstract:Abstract Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by Serpins in hemolymph. We recently isolated Manduca sexta Serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387–395). To further characterize its structure and function, we cloned Serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta Serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster Serpin-5, an immune-responsive protein. We produced Serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of Serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of Serpin-6 and PAP-3 is 2.6 × 104 m-1 s-1, indicating that Serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of Serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of Serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using Serpin-6 antibody. These results suggest that Serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.
Michael R. Kanost - One of the best experts on this subject based on the ideXlab platform.
-
inhibition of immune pathway initiating hemolymph protease 14 by manduca sexta Serpin 12 a conserved mechanism for the regulation of melanization and toll activation in insects
Insect Biochemistry and Molecular Biology, 2020Co-Authors: Yang Wang, Michael R. Kanost, Xiaolong Cao, Fan Yang, Rudan Huang, Susan M Paskewitz, Steve Hartson, Haobo JiangAbstract:A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by Serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta Serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with Serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against Serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal β-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with Serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by Serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae Serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a Serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.
-
manduca sexta Serpin 12 controls the prophenoloxidase activation system in larval hemolymph
Insect Biochemistry and Molecular Biology, 2018Co-Authors: Fan Yang, Michael R. Kanost, Xiaolong Cao, Yang Wang, Niranji Sumathipala, Haobo JiangAbstract:Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the Serpin superfamily. The enzyme system also leads to proteolytic processing of a Spatzle precursor. Binding of Spatzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spatzle to induce immunity-related genes. Here we provide evidence that M. sexta Serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other Serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to Serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The Serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the Serpin-12 core domain (Serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the Serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both Serpin-12 and HP14, suggesting that a covalent Serpin-protease complex formed when Serpin-12 inhibited HP14. Together, these data suggest that M. sexta Serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.
-
Manduca sexta Serpin-7, a putative regulator of hemolymph prophenoloxidase activation
Insect biochemistry and molecular biology, 2013Co-Authors: Chansak Suwanchaichinda, Rose Ochieng, Shufei Zhuang, Michael R. KanostAbstract:Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six Serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca Serpin, named Serpin-7. The open reading frame of Serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta Serpins indicated that Serpin-7 contains Arg–Ile at the position of the predicted scissile bond cleaved by protease in the Serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M. sexta Serpin-4 and Serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant Serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable Serpin-protease complex. Considering that Serpin-3 and Serpin-6 are also efficient inhibitors of PAP3, it appears that multiple Serpins present in plasma may have redundant or overlapping functions. We conclude that Serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.
-
manduca sexta Serpin 5 regulates prophenoloxidase activation and the toll signaling pathway by inhibiting hemolymph proteinase hp6
Insect Biochemistry and Molecular Biology, 2010Co-Authors: Michael R. KanostAbstract:Insect immune responses include prophenoloxidase (proPO) activation and Toll pathway initiation, which are mediated by serine proteinase cascades and regulated by Serpins. Manduca sexta hemolymph proteinase-6 (HP6) is a component of both pathways. It cleaves and activates proPO activating proteinase 1 (PAP1) and hemolymph proteinase-8 (HP8), which activates proSpatzle. Inhibitors of HP6 could have the capability of regulating both of these innate immune proteinase cascade pathways. Covalent complexes of HP6 with Serpin-4 and Serpin-5 were previously isolated from M. sexta plasma using immunoaffinity chromatography with Serpin antibodies. We investigated the inhibition of purified, recombinant HP6 by Serpin-4 and Serpin-5. Both Serpin-4 and Serpin-5 formed SDS-stable complexes with HP6 in vitro, and they inhibited the activation of proHP8 and proPAP1. Serpin-5 inhibited HP6 more efficiently than did Serpin-4. Injection of Serpin-5 into larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced the bacteria-induced expression of attacin, cecropin and hemolin genes in fat body. Injection of Serpin-4 had a weaker effect on antimicrobial peptide expression. These results indicate that Serpin-5 may regulate the activity of HP6 to modulate proPO activation and antimicrobial peptide production during immune responses of M. sexta.
-
manduca sexta Serpin 4 and Serpin 5 inhibit the prophenol oxidase activation pathway cdna cloning protein expression and characterization
Journal of Biological Chemistry, 2005Co-Authors: Youren Tong, Michael R. KanostAbstract:Infection stimulates the innate immune responses of insects, including activation of prophenol oxidase (pro-PO) in plasma as the last step of a serine protease cascade. To investigate the roles of protease inhibitors in regulating this pathway, we cloned cDNAs for two new Serpins (Serpin-4 and Serpin-5) from the tobacco hornworm, Manduca sexta. Serpin-4 and Serpin-5 mRNAs are constitutively expressed at a low level in larval hemocytes and fat body and increased dramatically upon bacterial challenge. These Serpins are present in larval plasma at ∼3 (Serpin-4) and ∼1 μg/ml (Serpin-5) and increased 3–8-fold by 24 h after injection of bacteria or fungi. Recombinant Serpin-4 and Serpin-5 decreased pro-PO activation when added to plasma, but they did not directly inhibit the pro-PO-activating proteases. Instead, they apparently regulate the pathway by inhibiting one or more target proteases upstream of the pro-PO-activating proteases.
James A Huntington - One of the best experts on this subject based on the ideXlab platform.
-
thrombin inhibition by Serpins disrupts exosite ii
Journal of Biological Chemistry, 2010Co-Authors: Daniel J D Johnson, Vincenzo De Filippis, Ty E Adams, Nicola Pozzi, James A HuntingtonAbstract:Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all Serpins, whose mechanism involves significant conformational change in both Serpin and protease. It has been shown that the formation of the thrombin-Serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by Serpin complexation. Here we investigate the conformation of thrombin in its final complex with Serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ′-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting Serpins. Our data suggest a novel framework for understanding Serpin function, especially with respect to thrombin inhibition, where Serpins functionally “rezymogenize” proteases to ensure complete loss of activity and cofactor binding.
-
crystal structure of a stable dimer reveals the molecular basis of Serpin polymerization
Nature, 2008Co-Authors: Masayuki Yamasaki, Daniel J D Johnson, James A HuntingtonAbstract:The Serpins are a family of proteins that can multimerize via β-sheet linkages. Accumulation of such multimers can give rise to diseases such as thrombosis, cirrhosis and dementia. While the structures of many Serpins are known, the structure of the linkage between monomers was unclear. In this work, Huntington and colleagues have solved the crystal structure of an antithrombin dimer. They find that the high stability of the Serpin polymer is due to a large domain swap between beta sheets of the neighbouring monomers. In addition, the structure explains the how certain pathogenic mutations stabilize a polymerogenic folding intermediate. Repeating intermolecular protein association by means of β-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer’s, Huntington’s and Parkinson’s and the prion encephalopathies1. A family of proteins, known as the Serpins, also forms large stable multimers by ordered β-sheet linkages leading to intracellular accretion and disease2. These ‘Serpinopathies’ include early-onset dementia caused by mutations in neuroSerpin, liver cirrhosis and emphysema caused by mutations in α1-antitrypsin (α1AT), and thrombosis caused by mutations in antithrombin3. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the β-sheet expansion disorders collectively known as the conformational diseases4. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable Serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel β-strands inserting in the centre of the principal β-sheet of the neighbouring monomer. This structure explains the extreme stability of Serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible β-sheet expansion.
-
Serpins in thrombosis hemostasis and fibrinolysis
Journal of Thrombosis and Haemostasis, 2007Co-Authors: Lea M Beaulieu, James A Huntington, Frank C ChurchAbstract:Summary. Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (Serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of Serpins, using therapeutics to manipulate the Serpin–protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of Serpins, their cofactors, and their structure–function relationships is imperative for the development of state-of-the-art pharmaceuticals for the selective fine-tuning of hemostasis and fibrinolysis. This review describes key Serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z-dependent protease inhibitor, α1-protease inhibitor, protein C inhibitor, α2-antiplasmin and plasminogen activator inhibitor-1. We focus on the biological function, the important structural elements, their known non-hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific Serpins.
-
homozygous deficiency of heparin cofactor ii relevance of p17 glutamate residue in Serpins relationship with conformational diseases and role in thrombosis
Circulation, 2004Co-Authors: Javier Corral, James A Huntington, Justo Aznar, Rocio Gonzalezconejero, Piedad Villa, Antonia Minano, Amparo Vaya, Robin W Carrell, Vicente VicenteAbstract:Background— Heparin cofactor II (HCII) is a hepatic Serpin with significant antithrombin activity that has been implicated in coagulation, inflammation, atherosclerosis, and wound repair. Recent data obtained in mice lacking HCII suggest that this Serpin might inhibit thrombosis in the arterial circulation. However, the clinical relevance and molecular mechanisms associated with deficiency of HCII in humans are unclear. Methods and Results— We studied the first family with homozygous HCII deficiency, identifying a Glu428Lys mutation affecting a conserved glutamate at the hinge (P17) of the reactive loop. No carrier reported arterial thrombosis, and only 1 homozygous HCII-deficient patient developed severe deep venous thrombosis, but she also had a de novo Glu100Stop nonsense truncation in the antithrombin gene. Conclusions— Our results confirm the key structural role of the P17 glutamate in Serpins. The same mutation causes conformational instability and polymerization in 3 Serpins: Drosophila necrotic, h...
-
mechanisms of glycosaminoglycan activation of the Serpins in hemostasis
Journal of Thrombosis and Haemostasis, 2003Co-Authors: James A HuntingtonAbstract:Summary. Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The Serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final Serpin–protease complex. The Serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several Serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and α2-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the Serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the Serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.
Robin W Carrell - One of the best experts on this subject based on the ideXlab platform.
-
structural mechanism for the carriage and release of thyroxine in the blood
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Aiwu Zhou, Randy J Read, Robin W CarrellAbstract:The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the Serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 A, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other Serpins in having the upper half of its main β-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other Serpins. Thus, TBG has adapted the Serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.
-
homozygous deficiency of heparin cofactor ii relevance of p17 glutamate residue in Serpins relationship with conformational diseases and role in thrombosis
Circulation, 2004Co-Authors: Javier Corral, James A Huntington, Justo Aznar, Rocio Gonzalezconejero, Piedad Villa, Antonia Minano, Amparo Vaya, Robin W Carrell, Vicente VicenteAbstract:Background— Heparin cofactor II (HCII) is a hepatic Serpin with significant antithrombin activity that has been implicated in coagulation, inflammation, atherosclerosis, and wound repair. Recent data obtained in mice lacking HCII suggest that this Serpin might inhibit thrombosis in the arterial circulation. However, the clinical relevance and molecular mechanisms associated with deficiency of HCII in humans are unclear. Methods and Results— We studied the first family with homozygous HCII deficiency, identifying a Glu428Lys mutation affecting a conserved glutamate at the hinge (P17) of the reactive loop. No carrier reported arterial thrombosis, and only 1 homozygous HCII-deficient patient developed severe deep venous thrombosis, but she also had a de novo Glu100Stop nonsense truncation in the antithrombin gene. Conclusions— Our results confirm the key structural role of the P17 glutamate in Serpins. The same mutation causes conformational instability and polymerization in 3 Serpins: Drosophila necrotic, h...
-
crystal structures of native and thrombin complexed heparin cofactor ii reveal a multistep allosteric mechanism
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Trevor Baglin, Frank C Church, Robin W Carrell, Charles T Esmon, James A HuntingtonAbstract:The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the Serpin family, which use a unique β-sheet expansion mechanism to trap and destroy their targets. Since the discovery that Serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid Serpin β-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of Serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin–HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central β-sheet of HCII.
-
structure of a Serpin protease complex shows inhibition by deformation
Nature, 2000Co-Authors: James A Huntington, Randy J Read, Robin W CarrellAbstract:The Serpins have evolved to be the predominant family of serine-protease inhibitors in man1,2. Their unique mechanism of inhibition involves a profound change in conformation3, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical Serpin–protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the Serpin. This cleaves the reactive centre, which then moves 71 A to the opposite pole of the Serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable Serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation4. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction5,6. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the Serpins with their selective advantage.
-
what do dysfunctional Serpins tell us about molecular mobility and disease
Nature Structural & Molecular Biology, 1995Co-Authors: Penelope E Stein, Robin W CarrellAbstract:Proteinase inhibitors of the Serpin family have a unique ability to regulate their activity by changing the conformation of their reactive-centre loop. Although this may explain their evolutionary success, the dependence of function on structural mobility makes the Serpins vulnerable to the effects of mutations. Here, we describe how studies of dysfunctional variants, together with crystal structures of Serpins in different forms, provide insights into the molecular functions and remarkable folding properties of this family. In particular, comparisons of variants affecting different Serpins allow us to define the domains which control this folding and show how spontaneous but inappropriate changes in conformation cause diverse diseases.
Yang Wang - One of the best experts on this subject based on the ideXlab platform.
-
inhibition of immune pathway initiating hemolymph protease 14 by manduca sexta Serpin 12 a conserved mechanism for the regulation of melanization and toll activation in insects
Insect Biochemistry and Molecular Biology, 2020Co-Authors: Yang Wang, Michael R. Kanost, Xiaolong Cao, Fan Yang, Rudan Huang, Susan M Paskewitz, Steve Hartson, Haobo JiangAbstract:A network of serine proteases (SPs) and their non-catalytic homologs (SPHs) activates prophenoloxidase (proPO), Toll pathway, and other insect immune responses. However, integration and conservation of the network and its control mechanisms have not yet been fully understood. Here we present evidence that these responses are initiated through a conserved serine protease and negatively regulated by Serpins in two species, Manduca sexta and Anopheles gambiae. We have shown that M. sexta Serpin-12 reduces the proteolytic activation of HP6, HP8, proPO activating proteases (PAPs), SPHs, and POs in larval hemolymph, and we hypothesized that these effects are due to the inhibition of the immune pathway-initiating protease HP14. To test whether these changes are due to HP14 inhibition, we isolated a covalent complex of HP14 with Serpin-12 from plasma using polyclonal antibodies against the HP14 protease domain or against Serpin-12, and confirmed formation of the complex by 2D-electrophoresis, immunoblotting, and mass spectrometry. Upon recognition of bacterial peptidoglycans or fungal β-1,3-glucan, the zymogen proHP14 became active HP14, which formed an SDS-stable complex with Serpin-12 in vitro. Activation of proHP21 by HP14 was suppressed by Serpin-12, consistent with the decrease in steps downstream of HP21, proteolytic activation of proPAP3, proSPH1/2 and proPO in hemolymph. Guided by the results of phylogenetic analysis, we cloned and expressed A. gambiae proSP217 (an ortholog of HP14) and core domains of A. gambiae Serpin-11 and -17. The recombinant SP217 zymogen became active during expression, with cleavage between Tyr394 and Ile395. Both MsHP14 and AgSP217 cleaved MsSerpin-12 and AgSRPN11 at Leu*Ser (P1*P1') and formed complexes in vitro. ProPO activation in M. sexta plasma increased after recombinant AgSP217 had been added, indicating that it may function in a similar manner as the endogenous initiating protease HP14. Based on these data, we propose that inhibition of an initiating modular protease by a Serpin may be a common mechanism in holometabolous insects to regulate proPO activation and other protease-induced immune responses.
-
manduca sexta Serpin 12 controls the prophenoloxidase activation system in larval hemolymph
Insect Biochemistry and Molecular Biology, 2018Co-Authors: Fan Yang, Michael R. Kanost, Xiaolong Cao, Yang Wang, Niranji Sumathipala, Haobo JiangAbstract:Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the Serpin superfamily. The enzyme system also leads to proteolytic processing of a Spatzle precursor. Binding of Spatzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spatzle to induce immunity-related genes. Here we provide evidence that M. sexta Serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other Serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to Serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The Serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the Serpin-12 core domain (Serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the Serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both Serpin-12 and HP14, suggesting that a covalent Serpin-protease complex formed when Serpin-12 inhibited HP14. Together, these data suggest that M. sexta Serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.
-
purification and characterization of manduca sexta Serpin 6 a serine proteinase inhibitor that selectively inhibits prophenoloxidase activating proteinase 3
Insect Biochemistry and Molecular Biology, 2004Co-Authors: Yang Wang, Haobo JiangAbstract:The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by Serpin-1J and Serpin-3. To test if other Serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta Serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the Serpin-1 or Serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of Serpin-6 were found to be 46,710±10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta Serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple Serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, Serpin-6 efficiently blocked the cleavage activation of proPO.
